There are >300 million cases and ~1 million deaths p.a. caused by Plasmodium falciparum (Pf) malaria. Control using bednets, insecticides, and drugs, has been significant but as stressed recently in The Lancet editorial, a malaria vaccine is needed. The ideal single stage vaccine would target the pre-erythrocytic (sporozoite (spz)/liver) stages to prevent infection (and disease and transmission) in >80% of recipients and would have huge public and private sector markets. Such high-level (>90%), sustained protection has been achieved only in volunteers immunized with radiation-attenuated Pf spz by the bites of infected mosquitoes. Sanaria, founded to develop such a vaccine, has manufactured a metabolically active, non-replicating attenuated Pf spz vaccine (the PfSPZ Vaccine), which fulfilled all FDA regulatory criteria. In a phase 1 clinical trial, PfSPZ Vaccine administered subcutaneously (SC) or intradermally (ID) was safe and well tolerated in all 80 healthy volunteers, induced specific antibody and T cell responses, but sterile protection in just two. Our next critical goal is to establish high-level protective efficacy. Recent experimental animal data have indicated that responses elicited by SC and ID immunizations maybe insufficient to reproducibly induce protection. It requires 7-10x more irradiated P. yoelii (irr Py) spz administered SC or ID compared to intravenously (IV) to achieve >80% protection in mice. IV immunization of rhesus monkeys with the PfSPZ Vaccine elicits 30-150x higher antibody and T cell responses compared to SC, and only IV immunization induces critical CD8+ T cells in the liver necessary for protection. Therefore, we aim to administer the vaccine IV in the next clinical trial to establish safety, demonstrate high protection, and develop assays that predict protection. We will then focus on reducing the number of doses IV, specifically targeting >80% protection with a single dose, and develop alternative parenteral methods of administration that duplicate the efficacy of IV administration. Such a vaccine would have a large market, especially in high-risk travelers, military from all parts of the world, and in geographically focused Pf elimination campaigns. Our data indicate that high level protection is achievable with a single dose of spz when they are administered with an adjuvant. A single dose IV of irrPy spz with 7DW8-5, an analogue of 1-galactosylceramide and a ligand for natural killer T cells, induces 70-90% protection. In 3 experiments 30/40 (75%) mice immunized with a single dose of irrPy spz+adjuvant, 0/15 with adjuvant alone, and 4/30 mice (13%) with irrPy spz alone were completely protected against virulent Py spz challenge. In this Phase I project we will use Py in mice to demonstrate that the number of doses and overall number of irrPy spz required to achieve >80% protection by the IV and non-IV parenteral routes can be significantly reduced by administering attenuated spz with 7DW8-5. We will 1) optimize the single dose IV/7DW8-5 regimen of irrPy spz, 2) reduce the number of irr Py spz per dose in a 3 dose IV/7DW8-5 regimen, 3) optimize non-IV routes to administer irrPy spz with 7DW8-5, 4) enhance immune responses in mice to PfSPZ Vaccine using 7DW8-5.